Imaging oxygen in vivo presents a challenging and important problem in modern physiology and medicine. A number of methods for mapping oxygenation in biological objects have been proposed in the past, but none of them provides means for microscopic imaging with three-dimensional capability. We propose to develop a technique for microscopic imaging of oxygen in biological systems by coupling the phosphorescence quenching method with two-photon laser scanning microscopy (2P LSM). The work plan has three main objectives: 1) synthetic development of two-photon excitable phosphorescent probes and their photophysical characterization;2) construction of two-photon imaging system for phosphorescence (long radiative lifetimes of phosphorescent probes make standard systems for two-photon imaging non- suitable for this application);3) testing the technique using biological systems for which information about the distribution of oxygen at microscopic (sub-cellular) level is of high significance. These systems are: oxygen gradients formed-during phagocytic endocytosis (S. Muro, Penn Pharmacology);oxygen gradient between the choroid and the retina of the eye (E. Pugh, Penn Ophthalmology);oxygen distribution in the brain (D. Boas, Harvard/MGH). Development of two-photon phosphorescent oxygen nanosensors is the key to the proposal, and the corresponding part of the work plan (Specific Aims 1 and 2) is a continuation of the pilot project funded by the NIH grant R21 EB-003663-01. The design of the sensor combines principles and approaches currently under scrutiny in several nanotechnology applications: two-photon absorption (2PA) by an antenna-array, consisting of multiple chromophores, coupled to the same functional core;intramolecular energy transfer (ET) from the antenna to the core;dendritic encapsulation of the core to achieve its protection and to control its local environment. The feasibility of the design has been demonstrated and the ways of optimization of probes for in vivo applications have been identified. Furthermore, the results obtained reveal changes that will have to be made to two-photon standard imaging setup to permit imaging of phosphorescence. The biological systems are selected to evaluate the performance of the technique and to simultaneously obtain valuable information for the related bio-medical problems.